Castanospermum australe A. Cunn. (Leguminosae), the Moreton Bay Chestnut or Black Bean, the monotypic species of the genus Castanospermum native to northeastern Australia, has been introduced into the Indian subcontinent, South Africa and mild climate areas of North America as an ornamental tree. (1) Castanospermine (FIG. 4) the major alkaloidal constituent of the toxic, chestnut-like seeds is a tetrahydroxyindolizidine alkaloid (2) having potent alpha- and beta-glucosidase inhibitory activity (3,4). The consequent modification of glycoprotein processing in cells due to inhibition of glucosidase I (5) ) has stimulated considerable interest in the biological effects of castanospermine on a variety of organisms. The alkaloid is an intensely active feeding deterrent and toxin to certain insects (6,7), and adversely affects root length growth in a number of dicotyledonous plants (8). It has been shown to alter glycogen metabolism and distribution (9) and to block the hyperglycemic response to carbohydrates (10) in rats. In addition, castanospermine inhibits replication of the human immunodeficiency virus (HIV) (11) and other retroviruses (12), and reduces tumor growth in mice (13). Furthermore, the structurally related trihydroxyindolizidine alkaloid swainsonine (FIG. 7), the toxic constituent of locoweeds (14) and Australian Swainsona species (15) is a powerful inhibitor of alpha-mannosidase and exhibits anti-metastatic immunomodulation towards melanoma cells in mice (16). Polyhydroxyindolizidine alkaloids and structurally related analogs may thus represent a class of alkaloids capable of profoundly influencing diverse biological processes due to their glycosidase inhibitory activity (17) and have stimulated efforts to isolate or synthesize additional compounds in order to delineate structure-activity relationships.
Among the common types of glycoproteins that are found in eukaryotic organisms, both as cell-associated proteins and as secreted proteins, are those having N-linked or asparagine-linked oligosaccharides (18; 19). The biosynthesis of the oligo-saccharide portion of these molecules involves a complex sequence of events beginning with the synthesis of the Glc.sub.3 Man.sub.9 (GlcNAc).sub.2 -pyrophosphoryl-dolichol intermediate and the transfer of the carbohydrate portion of this intermediate to various asparagine residues on the newly-synthesized polypeptide (20, 21, 22). Following the transfer of this oligosaccharide to the protein, the newly formed glycoprotein undergoes a number of modification or "processing" reactions which begin in the endoplasmic reticulum and continue as the glycoprotein is transported through the Golgi to its final destination (23, 24, 25, 26).
The initial processing reactions, catalyzed by two endoplastic reticulum membrane-bound glucosidases, involve the removal of the three glucose residues. Glucosidase I removes the outermost alpha-1, 2-linked glucose residue, while glucosidase II releases the remaining two alpha-1, 3-linked glucoses (27, 28, 29, 30, 31). These trimming reactions give rise to a Man.sub.9 (GlcNAc).sub.2 -protein which may be acted upon by an endoplasmic reticulum-bound alpha-1, 2-mannosidase to give a Man.sub.8 (GlcNAc).sub.2 -oligosaccharide structure (32, 33). The Man.sub.8-9 (GlcNAc).sub.2 -protein may be the direct precursor of the high-mannose glycoproteins, or it may be further processed, after translocation to the Golgi, to yield hybrid or complex types of glycoproteins.
In the Golgi, the Man.sub.8-9 (GlcNAc).sub.2 -protein may be the substrate for mannosidase I which removes the remaining alpha-1, 2-linked mannose residues, generating a Man.sub.5 (GlcNAc).sub.2 -protein (34, 35, 36, 37, 38). This glycoprotein can then serve as acceptor for GlcNAc transferase I which catalyzes the addition of a GlcNAc, from UDP-GlcNAc, to the mannose that is linked alpha-1, 3 to the beta-linked mannose, resulting in the formation of a GlcNAcMan.sub.5 (GlcNAc).sub.2 -protein (39, 40, 41). Following this addition a second Golgi mannosidase, mannosidase II, catalyzes the removal of the alpha-1, 3 and alpha-1, 6-linked mannose residues generating a GlcNAc-Man.sub.3 (GlcNAc).sub.2 -oligosaccharide (39, 42). Failure to remove these two terminal mannose units can result in the formation of hybrid types of glycoproteins (43, 22). The GlcNAc-Man.sub.3 (GlcNAc).sub.2 -protein can then be the substrate for a series of glycosyltransferases forming the complex types of glycoproteins that contain galactose, sialic acid, fucose, etc. (44, 25).
The study of the biosynthesis of the oligosaccharide portion of the N-linked glycoproteins has been greatly facilitated by the use of inhibitors that act at specific steps in the processing pathway (62, 45). Some of these . inhibitors which have gained popularity over the past several years include swainsonine (see FIG. 2), a Golgi mannosidase II inhibitor originally isolated from the Australian plant Swainsona canescens (15, 46, 47), deoxymannojirimycin, a synthetic Golgi mannosidase I inhibitor (48, 49, 64), and castanospermine, a glucosidase I/glucosidase II inhibitor isolated from the seeds of the Australian tree Castanospermum australe (2, 5, 50).